One of the long-term goals of the Ochman laboratory is to investigate factors underlying process of sequence evolution in bacteria. A basic premise of molecular evolutionary biology is that mutations occur at random throughout the genome and that variation in the pattern of nucleotide substitutions is the direct result of natural selection. However, several lines of evidence suggest that this is not the case. Mutations arise through errors during the DNA replication; and because the mechanisms for replicating the complementary strands differ, it is possible for the rates of mutations to differ on the two strands. In addition, there are differences in the frequencies of mutations of the coding and non-coding strands of DNA, suggesting that certain processes, such as selection on codon usage, deamination events and transcription- coupled repair, are targeted to one of the complementary strands. The goals of the proposed research are to determine if the leading and lagging strands of DNA have unequal mutation rates, and to ascertain the source of mutational symmetries in transcribed portions of the genome. These issues will be examined by two complementary approaches: an experimental assay of mutation rates in genes introduced into the chromosome in opposite orientations, and an analysis of substitutions in chromosomal regions that differ with respect to coding potential and level of expression. Cumulatively, these studies will establish the relative roles of replication errors and DNA damage in generating the naturally- occurring genetic variation observed in populations.